Diarrhea is the most common health problem among travellers visiting less developed or tropical countries [1,2]. Although a number of enteropathogens have been implicated in traveller's diarrhea, the most common microorganism associated with the disease is enterotoxigenic Escherichia coli (ETEC) which is responsible for over half of the reported cases [3]. ETEC isolates are also the causative agents for the majority of diarrheal cases in young children and infants in developing tropical countries [4]. In addition, diarrhea caused by ETEC is an important concern for military personnel when deployed to less developed countries [5,6].
ETEC isolates that cause diarrhea have several virulence factors that play important roles in the disease process. They include two enterotoxins, heat-labile toxin (LT) and heat-stable toxin (ST) and bacterial surface adhesins called pili which allow the organism to colonize the intestinal tract. Both toxins are not required to cause diarrhea. Some clinical ETEC isolates have been shown to produce either LT or ST, while other isolates have both toxins. Strains that possess LT tend to be associated with more severe cases of traveller's diarrhea, while ETEC strains that produce only ST cause milder diarrhea.
Of the three factors produced by ETEC that are implicated in causing diarrhea, two are mediated by a specific interaction with a cell surface oligosaccharide receptor. The enterotoxin LT utilizes the ganglioside GM1 (.beta.Gal(1-3).beta.GalNAc(1-4)[.alpha.NeuAc(2-3)].beta.Gal (1-4).beta.Glc-ceramide) to bind to host cells and induce diarrhea by stimulating adenylate cyclase activity [7,8]. Two types of pili are found in the outer membranes of ETEC. The most important type of pili associated with pathogenic E. coli strains are called colonization factor antigens (CFA) or coli surface antigens (CS) which are responsible for allowing the organism to colonize the intestinal mucosa. Several potential oligosaccharide receptors have been identified for CFA and include the asialo GM1 glycolipid structure (.beta.Gal(1-3).beta.GalNAc(1-4).beta.Gal(1-4).beta.Glc-ceramide) as well as several sialic acid containing glycoconjugates [9,10]. In addition, the GalNAc(1-4)Gal disaccharide sequence has been shown to be a binding sequence for enterotoxigenic E. coli that express CS3 pili [11]. The other pili, type 1, are commonly found in E. coli strains, but do not appear to play a major role in causing diarrhea. Type 1 pili utilize mannose-containing oligosaccharide structures as a receptor. The other toxin associated with ETEC infections, ST, is a small polypeptide that interacts with its host cell receptor via a protein-protein interaction and induces diarrhea by increasing the levels of cyclic GMP in cells.
The current therapy for traveller's diarrhea is to initiate treatment with agents such as bismuth subsalicylate, Loperamide or agents such as Kaopectate in combination with rehydration therapy. The majority of the treatments involve the non-specific removal of the offending agents (i.e. toxins) from the intestinal tract. Only in moderate to severe cases of diarrhea where distressing or incapacitating symptoms are reported is antimicrobial therapy recommended. Antibiotics are not usually effective at reducing clinical symptoms of the disease and problems associated with antibiotic resistance can occur. A therapy is needed which would involve the specific removal of enterotoxigenic E. coli and/or LT activity from the intestine. This would lead to more rapid recovery and/or the lessening of symptoms in individuals who are suffering from diarrhea.
E. coli heat-labile enterotoxin (LT) has been found to display a lectin-like activity which allows it to bind to an oligosaccharide receptor on epithelial cells. Several oligosaccharide sequences have been identified as potential receptors for LT. Several glycolipids and their derivatives can serve as receptors for LT and include GM1 (.beta.Gal(1-3).beta.GalNAc(1-4)[.alpha.NeuAc(2-3)].beta.Gal(1-4).beta.Glc -ceramide). Other gangliosides which have been shown to bind LT include [12,13]GD1b (.beta.Gal(1-3).beta.GalNAc(1-4)[.alpha.NeuAc(2-3).alpha.NeuAc(2-3)].beta. Gal(1-4).beta.Glc-ceramide) and GM2 (.beta.GalNAc(1-4)[.alpha.NeuAc(2-3)].beta.Gal(1-4).beta.Glc-ceramide). Other derivatives of the ganglioside GM1 that were shown to bind LT include [12]: .beta.Gal(1-3).beta.GalNH2(1-4)[.alpha.Neu-NH2(2-3)].beta.Gal(1-4).beta.Gl c-ceramide; .beta.Gal(1-3).beta.GalNAc(1-4)[.alpha.NeuAcR(2-3)].beta.Gal(1-4).beta.Glc -ceramide, where R is the methyl ester of sialic acid; .beta.Gal(1-3).beta.GalNAc(1-4)[.alpha.(C7)NeuAc(2-3)].beta.Gal(1-4).beta. Glc-ceramide; and .beta.Gal(1-3).beta.GalNAc(1-4)[.alpha.NeuAcR(2-3)].beta.Gal(1-4).beta.Glc -ceramide, where R is ethanolamineamide.
In addition to glycolipid receptors, LT can utilize glycoproteins as receptors for toxin binding. LT has been shown to utilize glycoproteins that terminate in polylactosamine (.beta.Gal(1-4).beta.GlcNAc-) sequences [7]. LT also has the capability to bind to glycoproteins that terminate in lactose (.beta.Gal(1-4).beta.Glc) [14-16].
In addition, highly purified LT preparations have been obtained using galactose affinity columns [17].
In view of the above, there is a need for a compound which would treat traveller's diarrhea. A preferred compound would be administered noninvasively, such as orally, and would specifically remove toxin and/or organisms from the intestinal tract.